Many drugs have to be administered by injection since they are either degraded or absorbed inefficiently when given for instance orally or nasally or by the rectal route. A drug formulation intended for parenteral use has to meet a number of requirements in order to be approved by the regulatory authorities for use in humans. Thus, it has to be biocompatible and biodegradable and all substances used and their degradation products should be non toxic. In addition thereto, particulate drugs intended for injection have to be small enough to pass through the injection needle, which preferably means that they should be smaller than 200 .mu.m. The drug should not be degraded to any large extent in the formulation during production or storage thereof or after administration and should be released in a biologically active form with reproducible kinetics.
One class of polymers which fulfils the requirements as to biocompatibility and biodegradation to harmless end products are the linear polyesters based on lactic acid, glycolic acid and mixtures thereof. In the text below said polymers will also be referred to as PLGA. PLGA is degraded by ester hydrolysis to lactic acid and glycolic acid and has been shown to display excellent biocompatiblity. The innocous nature of PLGA is furthermore exemplified by the approval of several parenteral sustained release formulations based on these polymers by regulatory authorities, like the US Food and Drug Administration.
Parenterally administrable sustained release products on the market today based on PLGA include Decapeptyl.TM. (Ibsen Biotech), Prostap SR.upsilon. (Lederle), Decapeptyl.RTM. Depot (Ferring) och Zoladex.RTM. (Zeneca). The drugs of these formulations are all peptides. In other words they consist of amino acids condensed to a polymer with a relatively low degree of polymerisation and they do not have any well defined three-dimensional structure. This in turn generally permits the use of rather harsh conditions during preparations of said products. For example extrusion and subsequent size reduction can be used, which techniques should not be permissible in connection with proteins since they generally do not withstand such harsh conditions.
Consequently there is also a need for sustained release formulations for proteins. Proteins are similar to peptides in that they also consist of amino acids, but the molecules are larger and most proteins are dependant on a well defined three-dimensional structure as to many of their properties, including biological activities and immunogenicity. Their three-dimensional structures can relatively easily be destroyed, for example by high temperatures, surface induced denaturation and, in many cases exposure to organic solvents. Thus, a very serious drawback in connection with the use of PLGA, which is an excellent material per se, for sustained release of proteins is the requirement to utilize organic solvents to dissolve said PLGA, with the associated risk of compromising the stability of the protein.
Despite large efforts aiming at a modification of the PLGA technology in order to avoid this inherent problem with protein instability during the preparation process the progress in this field has been very slow and as yet no protein products have appeared on the market based on PLGA technology. The main reason therefore probably is that the three-dimensional structures of most proteins are too sensitive to withstand the preparation procedures used and/or being stored in a PLGA-matrix.
The most commonly used technique at present for entrapping water soluble substances such as proteins and peptides is the use of multiple emulsion systems. The drug substance is dissolved in a water or buffer solution and then mixed with an organic solvent, immiscible with water, containing the dissolved polymer. An emulsion is created having the water phase as the inner phase. Different types of emulsifiers and vigorous mixing are often used to create this first emulsion. Said emulsion is then transferred, under stirring, to another liquid, typically water, containing another polymer, for example polyvinylalalcohol, giving a triple w/o/w-emulsion. The microspheres are then hardened in some way. The most commonly used way is to utilize an organic solvent having a low boiling point, typically dichloromethane, and to evaporate the solvent. If the organic solvent is not fully immiscible with water, a continuous extraction procedure can be used by adding more water to the triple emulsion. A number of variations of this general procedure are also described in the literature. In some cases the primary emulsion is mixed with a non-aqueous phase, for instance silicon oil. Solid drug materials rather than dissolved drugs can also be used.
The release profiles of proteins from microspheres prepared by said method often show a fast initial release followed by a slower phase. Said slower phase can be followed by a third phase of faster release.
PLGA microspheres containing proteins are disclosed in WO-A1-9013780, the main feature of which is the use of very low temperatures during the manufacture of the microspheres in order to retain high biological activity of the proteins. The activity-of encapsulated superoxide dismutase was measured but merely on the portion released from the particles. This method has been used to produce PLGA microspheres containing human growth hormone in WO-A1-94l2l58 by dispersing human growth hormone in methylene chloride containing PLGA, spraying the obtained dispersion into a container with frozen ethanol with a layer of liquid nitrogen thereabove in order to freeze the droplets and allow them to settle in the nitrogen on to the ethanol. The ethanol is then thawed and the microspheres start to sink in the ethanol where the methylene chloride is extracted into the ethanol and the microspheres are hardened. This approach may be able to retain the stability of proteins better than most other processes for entrapping proteins in PLGA microspheres. However, this still remains to be unequivocally demonstrated for other proteins.
However, in the earlier mentioned methods based on encapsulation with PLGA the active substances are subjected to an organic solvent and this is generally detrimental to the stability of a protein. In addition thereto, the emulsion processes referred to above are complicated and likely to be problematic to scale up to an industrial scale. Furthermore, many of the organic solvents used in many of these processes are fraught with environmental problems and their high affinities for the PLGA polymer make removal difficult.
A parenterally administrable sustained release formulation should be able to control the release of the entrapped drug in an accurate way. In many of the systems based on PLGA the release of the active ingredient is largely dependent on the amount of drug substance incorporated into the microparticle, due to the formation of channels in the microparticles at higher drug loadings. This also contributes to a high initial burst at high drug loading.
A well known way of controlling the release of small molecules from a solid core is to apply a coating that produces a rate controlling film on the surface of the core. This is a general method of controlling the release rate of drugs to be administered by the oral route. One way of applying similar coats is by the use of air suspension technology. However, in connection with coating particles for use in parenteral administration, which particles are generally of a size below 200 .mu.m, and often smaller, generally severe problems are encountered. Such problems can be an increased tendency for particles to agglomerate and problems with static electricity disturbing the manufacturing process.
Some different ways of coating particles of such small sizes are dispersion of the drug in a solution of the coating material and subsequent spray drying and a number of coacervation methods where a dissolved polymer is used to encapsulate the core material in different ways. However, all these methods would expose a protein to the organic solvent used to dissolve the PLGA. A method where a fluidized bed is used in the coating of microparticles is disclosed in U.S. Pat. No. 4,568,559. Here a solid, dry composite admixture is prepared from a uniform dispersion of an active ingredient of a film-forming polymer, the admixture then being ground and the resulting particles being sieved to obtain a size distribution of 1-150 .mu.m. The core particles are then coated in a fluidized bed, a prerequisite, however, being that the same, or substantially the same, film-forming polymer material is used both for the preparation of the composite core and the coating to provide for bonding of the wall coating of the film-forming polymer to the core material. Thus, this method does not either eliminate the problem of exposing the protein to organic solvents if the film-forming polymer is PLGA or any other polymer that is not water soluble.
Thus, a method of producing parenterally administrable sustained release formulations for sensitive substances, for instance proteins, with the following properties would be highly desirable:
that can control the release rate of the entrapped substances within wide margins, typically from one or a few days to at least around one month; PA1 that enables the production to be carried out with standard pharmaceutical equipment and which can be used from small scale manufacture to full scale production; PA1 that makes it possible to eliminate, or minimise, the exposure of the active ingredient to organic solvents; and PA1 that is completely biodegradable and has a surface of a biocompatible material. PA1 2. Heat the suspension until the starch has been totally dissolved. PA1 3. Cool the solution to 50.degree. C. PA1 4. Add 96 ml of a 9,26% BSA solution (room temperature) in 50 mM sodium bicarbonate buffer pH 9,8 and stir for 10 seconds. PA1 5. Add starch-protein solution to 800 ml of a 20 w/w % polyethylene glycol solution in 50 mM sodium bicarbonate buffer pH 9,8 (room temperature, Av. Mol. Wt. 20000), under continous stirring. PA1 6. After 2 minutes,. add 3200 ml of a 40 w/w % polyethylene glycol solution in 50 mM sodium bicarbonate buffer pH 9,8 (room temperature, Av. Mol. Wt. 20000), under continous stirring. PA1 7. Stir for 24 h. PA1 8. The obtained microparticles are washed and vaccum dried. PA1 9. The dry microparticles are sieved through a 160 .mu.m mesh. PA1 1. Weigh out 80 g of starch (Amioca 50, National Starch) and suspend in 420 g of water. PA1 2. Heat the suspension until the starch has been totally dissolved. PA1 3. Cool the solution to 50.degree. C. PA1 4. Add the starch solution to 800 ml of a 20 w/w % polyethylene glycol solution in water (room temperature, Av. Mol. Wt. 20000 D), under continous stirring. PA1 5. After 2 minutes, add 3200 ml of a 40 w/w % polyethylene glycol solution in water (room temperature, Av. Mol. Wt. 20000 D), under continous stirring. PA1 6. Stir for 24 h. PA1 7. The obtained microparticles are washed and vacuum dried. PA1 8. The dried microparticles are impregnated with a 5% (w/w) BSA solution in water. Equal weight of particles and BSA-solution are used. PA1 9. After 3 h the particles are freeze dried. PA1 10.The dried microspheres are sieved through a 160 .mu.m sieve. PA1 1. Weigh out 80 g of starch (Amioca 50, National Starch) and suspend in 320 g of 50 mM sodium bicarbonate buffer pH 9,8. PA1 2. Heat the suspension until the starch has been totally dissolved. PA1 3. Cool the solution to 50.degree. C. PA1 4. Centrifuge 2511 ml of Monotard.RTM. from Novo Nordisk corresponding to 8.89 g insulin. Wash the insulin once with 500 ml of a buffer containing 0.15 M NaCl, 1 mM ZnCl.sub.2 and 10 mM Sodium acetate with a pH of 7.3 and centrifuge again. Mix the insulin with the starch solution and stir for 10 seconds. PA1 5. Add starch-protein solution to 800 ml of a 20 w/w % polyethylene glycol solution in 50 mM sodium bicarbonate buffer pH 9,8 (room temperature, Av. Mol. Wt. 20000), under continous stirring. PA1 6. After 2 minutes, add 3200 ml of a 40 w/w % polyethylene glycol solution in 50 mM sodium bicarbonate buffer pH 9,8 (room temperature, Av. Mol. Wt. 20000), under continous stirring. PA1 7. Stir for 24 h. PA1 8. The obtained microparticles are washed and vaccum dried. PA1 1. Weigh out 200 g of poly(lactide-co-glycolide 75/25) Resomer RG756 from Boeringer Ingelheim. PA1 2. Add 10 g of triacetin. PA1 3. Dissolve it in 3123 g of acetone. PA1 1. 500 g of starch microparticles containing 3,5% BSA are loaded in a Glatt GPCG 6" Wurster. PA1 2. The following conditions of the Wurster are set: PA1 1. Weigh out 200 g of poly(lactide-co-glycolide 50/50) Resomer RG504H from Boeringer Ingelheim. PA1 2. Dissolve it in 3133 g of acetone. PA1 1. 500 g of starch microparticles containing 3,5% BSA are loaded in a Glatt GPCG 6" Wurster. PA1 2. The following conditions of the Wurster are set: PA1 3. Recover the coated product PA1 1. Weigh out 40 g of poly(D,L lactide) Resomer R104 and 40 g of poly(lactide-co-glycolide 75/25) Resomer RG756 from Boeringer Ingelheim. PA1 2. Dissolve it in 1252 g of ethyl acetate. PA1 3. Mix 2504 g of water with 1,6 g of Tween 80. PA1 4. Mix the polymer solution and the water solution using an Ystral turrax mixer at high speed. PA1 1. 100 g of starch microparticles containing 2,7% BSA are loaded in a Huttlin Kugelcoater HKC005. PA1 2. The following conditions for the kugelcoater are set: PA1 3. After coating with PLGA, 200 g of a water solution containing 10 w/w % mannitol and 0,4 w/w % Tween 80 are sprayed onto the particles with a flow of 3,5 g/min. PA1 4. Recover the coated product.